Our past studies as well as those of others have indicated that alcohol abuse leads to a loss of docosahexaenoate (DHA), the major polyunsaturate in the nervous system. Nutritional inadequacies, particularly during early development, may also lead to such losses in this essential fatty acid. In following up this work, it is important to establish what losses in physiological functions are caused by the loss of DHA in various organ systems. [unreadable] [unreadable] In a collaboration with several investigators at Wayne State University, the relationships of alcohol intake during pregnancy is related to the mother's and newborn infant's essential fatty acid and vitamin status. Dietary intake of DHA throughout the study was estimated at 68 mg/day and might not support optimal fetal DHA accretion and subsequent neural development.[unreadable] [unreadable] The proportion of drinking days at the first prenatal visit is associated with decreased DHA in plasma and erythrocytes throughout the study and is the strongest at 24 weeks of gestation. In daily drinkers, high intakes of alcohol are associated with decreased DHA and arachidonic acid (AA) concentrations in plasma. The present findings indicate that maternal DHA deficiency is associated with high risk for fetal alcohol syndrome and may contribute to the mechanism(s) of alcohol-related neurodevelopmental disorders. Maternal folate was positively correlated with alcohol intake per drinking day, while infant venous plasma folate was negatively correlated with maternal smoking. It appears that concentrative transport of folate across the placenta occurs during pregnancy that smoking may negatively impact.[unreadable] [unreadable] Essential fatty acid metabolism was studied in male and female adults, both smokers and non-smokers, as a reference point for smoking alcoholics. Metabolism of D5-18:2n-6 and D5-18:3n-3 was studied in vivo after a single oral dose of the isotope mixture. Our results indicated that female smokers had a two-fold greater percent of the dose in their plasma, and a higher fractional rate for formation of D5-22:6n-3 from D5-22:5n-3 compared with non-smokers. Male smokers had elevated levels of total plasma n-3 fatty acids, more rapid turn over of 18:3n-3, a disappearance rate for D5-20:5n-3 that was both delayed and slower, and a greater percentage of D5-20:5n-3 that was directed towards D5-22:5n-3 formation relative to non-smokers. Smoking generally increased the bioavailability of plasma n-3 fatty acids, accelerated fractional synthetic rates, and increased the percent formation of some long chain n-3 polyunsaturated fatty acids. [unreadable] [unreadable] The relationship of dietary alcohol and essential fatty acid intakes were studied in 4168 adults taken from the cross-sectional National Health and Nutrition Examination Survey 2001-2002. Our results indicated that among men, decreased nutrient densities of saturated, monounsaturated, polyunsaturated, linoleic and alpha-linolenic acid were associated with alcohol consumption. Binge drinking men had significantly decreased intakes of saturated, monounsaturated, polyunsaturated and linoleic, alpha-linolenic, eicosapentaenoic and docosahexaenoic acids. Thus it appears that drinking alcohol lowers highly unsaturated fatty acids in tissues thru altered fatty acid metabolism but also thru altered food selection and dietary habits. [unreadable] [unreadable] Great progress was made in the successful rearing of pups from the second day of life using feeding bottles developed by Prof. J. Hoshiba. Rats and mice were raised on n-3 supplemeented or deficient milks to adulthood and tested in several behavioral tasks. There was no difference in motor activity or in the plus maze but escape latency and memory retention were poorer in the n-3 deficient rats. This technique now makes possible many experiments where control of individual fatty acids or other nutrients in the diet and thus in tissue composition is required. In the first such application, a major expperiment was performed where DPAn6 was compared to DHA feeding for the first 10 weeks of life. It was shown that the DPAn6-fed rats had the same deficit in spatial task performance as did the LA-fed group, i.e., that DPAn6 conferred none of the benefit that adding DHA did. Mice were also raised on similar diets by artificial rearing and a 50% loss of brain DHA was observed at adulthood in the n-3 deficient group. In the open field test, habituation was slower and decreases in the general level of activity were observed in the n-3 deficient group. Under usual conditions, no differences could be seen in the plus maze between these dietary fgroups. However, under stressful conditions, when a bright light and a loud noise were given during the testing, the n-3 deficient group spent less time in the open arms and made fewer entries and had fewer head dips, indicating a higher level of anxiety. The low DHA mice also performed less well in another spatial task, the reference memory version of the Barnes maze, but there were no differences observed in either the cued or the working memory versions. These experiments have established that the nervous sytem has a requirement for the 22-carbon n-3 highly unsaturated fatty acids for optimal function. [unreadable] [unreadable] In another major line of research, a two generational model of DHA deficiency in rats was used in order to characterize the loss in nervous system function. A deficit in spatial task peformance was observed in n-3 deficient rats using the Barnes circular maze along with a 58% loss of brain DHA. No differnces were observed in memory retention for this task but the n-3 adequate rats perfomed better in a reversal learning task. There was no difference in locomotor activity but slower habituation was observed in the open field apparatus. No differences between groups were observed in the plus maze.[unreadable] [unreadable] This two generational dietary model has been employed to produce mice on four different diets varying LNA and DHA so that four different levels of brain DHA are obtained. At adulthood, the animals were tested for prepulse inhibition (PPI), a measure of neuromotor gating. It was observed that four different levels of PPI was obtained in the four dietary groups with the most PPI being obtained in those with the highest DHA level, ie., those fed preformed DHA. A significantly lower PPI was observed in animals fed a high level of LNA but without preformed DHA even though it resulted in only about a 10% loss of brain DHA. This was the first experiment where it could be shown that there was a brain functional loss in animals fed LNA but without preformed DHA.[unreadable] [unreadable] A methodological development has been completed facilitating lipidomic and metabolomic approaches has been made with regard to high throughput fatty acid analysis. Labor intensive transmethylation procedures were simplified and then adopted to robotics. A robotic program and procedures, together with custom hardware have been developed and validated for plasma samples that can potentially produce 400 methyl ester samples per day. GCs have been converted to fast GC mode and analyses can now be completed within about 15 minutes. A program has been developed to process GC data for peak assignment and quantification. Large clinical studies are currently underway with this system. Umbilical cord samples from 2,500 subjects in the ALSPAC longitudinal cohort will be assayed. Additionally, 4,500 samples from 7 year olds will be assayed. In this cohort, trace metal analyses for methyl mercury and other elements is progressing. The benificial levels of essential fatty acids will be compared to the risks of these neurotoxicants.